High frequency electrical stimulation reduces α-synuclein levels and α-synuclein-mediated autophagy dysfunction

Accumulation of α-synuclein (α-Syn) has been implicated in proteasome and autophagy dysfunction in Parkinson’s disease (PD). High frequency electrical stimulation (HFS) mimicking clinical parameters used for deep brain stimulation (DBS) in vitro or DBS in vivo in preclinical models of PD have been found to reduce levels of α-Syn and, in certain cases, provide possible neuroprotection. However, the mechanisms by which this reduction in α-Syn improves cellular dysfunction associated with α-Syn accumulation remains elusive. Using HFS parameters that recapitulate DBS in vitro, we found that HFS led to a reduction of mutant α-Syn and thereby limited proteasome and autophagy impairments due to α-Syn. Additionally, we observed that HFS modulates via the ATP6V0C subunit of V-ATPase and mitigates α-Syn mediated autophagic dysfunction. This study highlights a role for autophagy in reduction of α-Syn due to HFS which may prove to be a viable approach to decrease pathological protein accumulation in neurodegeneration.


Primary neuron culture and AAV transduction
Pregnant Sprague-Dawley rats (E17) were purchased from Envigo.Embryos were surgically removed, and cortices were dissected in Hanks Balanced salt solution (Gibco).The meninges were removed, and cells were dissociated using a papain dissociation system (Worthington) before being resuspended in Neurobasal medium A supplemented with antibiotic-antimycotic solution (Gibco), L-glutamine substitute (GlutaMAX™; Gibco), and factor B27 (Gibco).
Neurons were plated on poly-D-lysine coated glass coverslips at a density of 5 × 10 5 cells/well or on poly-Dlysine coated 6-well cell culture plates at a density of 2 × 10 6 cells/well and incubated at 37 °C in 5% CO 2 with half media changes every 3 days.Neurons were transduced with the experimental AAVs (AAV-A53T, AAV-EV, AAV-YFP, AAV-mito-QC) 2 days post-isolation at a multiplicity of infection of 3000.Media containing AAV vectors were removed after 72 h and replaced with fresh media 5 days post-isolation and stimulated.Following stimulation protocol (see below), neurons were immediately fixed with 4% PFA for immunofluorescence staining or lysed for immunoblotting.

High frequency electrical stimulation
Transfected SH-SY5Y cells and transduced primary cortical neurons were stimulated using an IonOptix C-Pace EM multichannel stimulator configured with a 6-well C-Dish fitted with carbon electrodes.An external signal was generated using a waveform generator (Agilent 33220A, Keysight Technologies) with the following parameters: 120 Hz, 5 V, 0.4 ms pulse width, for a duration of 2 h.HFS of neurons was initiated in fresh media immediately following 72 h of AAV treatment on DIV5 (days in vitro) and fixed in 4% PFA at the end of the 2-h stimulation session.All immunofluorescence outcomes secondary to HFS were generated from 3 independent cell dissociations (n = 3) with 10 neurons or cells analysed per experiment.SH-SY5Y cells and cortical neurons were placed in the cell culture incubator during stimulation, with the IonOptix system placed outside the incubator.
www.nature.com/scientificreports/Quantitative real-time PCR was performed in the Quant Studio™ 5 Real-Time PCR System (ThermoFisher Scientific) using Power Track™ SYBR Green Master Mix (Invitrogen, Cat# A46109).The thermal cycling conditions were as follows: amplifications were performed starting at 95 °C for 2 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min.Melting curve analysis began at 95 °C for 15 s, followed by at 60 °C for 1 min and at 95 °C for 15 s.Specificity of the product amplification was confirmed by melting curve analysis.Primers for the target human gene α-Syn were forward-5'-CCA AAG AGC AAG TGA CAA ATG TTG -3' , reverse-5'-CCT CCA CTG TCT TCT GGG CTACT -3' .Primers for the house keeping human gene GAPDH were forward-5'-GTC TTC ACC ACC ATG GAG AA -3' , reverse-5'-ATC CAC AGT CTT CTG GGT GG -3' .

Immunofluorescence staining of cultured cells and neurons
Post fixation using 4% PFA, cells and neurons were permeabilized with 0.2% Triton X-100 for 15 min, washed three times with PBS and then incubated with blocking solution (1% BSA, 22.52 mg/mL glycine, 0.1% Tween-20 in PBS) for one hour, followed by overnight incubation with primary antibodies diluted in blocking solution at 4 °C.Primary antibodies were washed off with PBS, and cells were incubated in secondary antibodies diluted in blocking solution.Secondary antibodies were washed off with PBS.The coverslips were mounted on slides using ProLong TM Gold antifade Mountant with DAPI (ThermoFisher).

Western blot analysis
Cell lysates were scraped and pelleted by centrifugation at 2350 × g for 5 min at 4 °C.The supernatant was discarded, and the pellet was lysed in RIPA buffer (50 mM Tris-HCl pH 7.4, 1% Nonidet P-40, 150 mM NaCl, 1 mM EDTA) supplemented with protease (cOmplete™ ULTRA Tablets, Mini, EASYpack Protease Inhibitor Cocktail, Roche) and phosphatase (PhosSTOP EASYpack, phosphatase inhibitor tablets, Roche) inhibitors and kept on ice for 30 min.Following incubation, samples were centrifuged at 17,100 × g for 20 min at 4 °C.The insoluble pellet was discarded, and the supernatant was collected for subsequent analysis.Protein concentration in RIPAsoluble samples was quantified using the DC protein assay (BioRad).4X sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer was added to the whole cell lysate and boiled for 10 min at 95 °C.Samples were subsequently run on a 4-20% Mini-Protean TGX Precast gel (Bio-Rad).The gel was transferred onto polyvinylidene fluoride membrane (Bio-Rad) and blocked at room temperature in 5% w/v milk in 0.1% TBS-T for 1 h and incubated in primary antibodies overnight at 4 °C.The PVDF membrane was washed the next day with PBS-T and incubated in HRP-conjugated secondary antibodies and detected using enhanced chemiluminescence (Pierce).

Imaging and quantification
Confocal images were captured with a Zeiss LSM880 confocal microscope.SH-SY5Y cells and cortical neurons were imaged at 63X magnification with additional 2X zoom at 405 nm, 488 nm, 555 nm, and 639 nm laser lines.Z-stacks were taken within linear range at a constant gain for each channel at a 920 × 920 pixel-ratio.The software was programmed to acquire an image every ~ 0.5 μm, capturing all the cells visible in the z-plane in each field of view using a 63X oil objective.Fluorescence intensity was calculated using the Surface module, while puncta size was calculated using the Spots module in IMARIS (Oxford instruments).For quantification of the number of mito-lysosomes per cell and neuron, contours were manually drawn around the cell body and processes of SH-SY5Y cells and cortical neurons to define the region of interest (ROI).Using the IMARIS spots module, puncta were selected in both the mCherry and EGFP channels at an approximate size of 0.4 μm diameter.The 'co-localize spots' function was used to measure the number of mCherry puncta that were not co-localized with EGFP puncta.For quantification of the percentage of LC3B co-localized with TOMM20, contours were manually drawn around cells to define the ROI.Using IMARIS, the spots module was used to identify LC3B, and the surfaces module was used to identify TOMM20.The 'spots close to surface' function was used to measure the number of LC3B puncta that were and were not co-localized with TOMM20.The percentage of LC3B co-localized with TOMM20 was calculated by dividing the number of co-localized LC3B spots by the total number of LC3B spots.

Statistical analysis
Statistical analysis was performed using GraphPad Prism 8.The specific statistical tests performed are indicated.All data are represented as mean ± s.e.m. with 3 independent experiments.One-way ANOVA or Student t-test was performed as indicated.

High frequency electrical stimulation causes transient reduction in mutant α-Syn levels
We previously demonstrated that HFS reduced pathological α-Syn levels in cultured neurons 32 .Here we aimed to determine if similar modulation can be achieved in other cell types to facilitate dissection of the underlying mechanisms.To achieve this, we used human SH-SY5Y cells, an immortalized cell line routinely used in cell work for PD research 35,36 .SH-SY5Y cells were co-transfected with human A53T α-Syn or pcDNA with green fluorescent protein (GFP) as an internal control to assess for non-specific effects of HFS.We electrically stimulated the cells for 2 h using the IonOptix C-Pace EM Culture Pacing System with settings mimicking clinical DBS 32 and fixed immediately for immunofluorescent analysis (Fig. 1a).We observed that HFS did not alter levels of endogenous α-Syn based on relative fluorescence intensity (RFU) assessment (3.1 ± 0.27 RFU stimulated vs 3.2 ± 0.3 RFU non-stimulated).In contrast, HFS led to a partial reduction of mutant α-Syn in SH-SY5Y cells transfected with A53T α-Syn compared to non-stimulated cells transfected with A53T α-Syn (61.4 ± 5.67 RFU stimulated vs 150.8 ± 8.2 RFU unstimulated, ****p < 0.0001, one-way ANOVA, Tukey's m.c.t.) (Fig. 1b).This decrease was still significantly higher than stimulated pcDNA-transfected control cells (3.1 ± 0.27 RFU) (Fig. 1b).No differences in GFP fluorescence intensity were noted (109.1 ± 7.45 RFU stimulated vs 96.2 ± 7.12 RFU non-stimulated) (Fig. 1c), demonstrating the specificity of HFS on mutant α-Syn levels.We also found that stimulated and nonstimulated A53T α-Syn transfected cells exhibited similar α-Syn mRNA levels (1.07 ± 0.15 relative mRNA levels stimulated vs 1.115 ± 0.42 relative mRNA levels non-stimulated) (Fig. 1d), indicating that the decreased α-Syn levels were not caused by HFS modulation of α-Syn transcription but due to the alteration of protein levels.Taken together our results suggest that HFS does not inhibit the expression of α-Syn but promotes the degradation of accumulated protein.

α-Syn reduction due to high frequency electrical stimulation limits UPS dysfunction
The ubiquitin proteosome pathway mediates covalent tagging of multiple ubiquitin molecules to protein substrates, followed by degradation of the tagged proteins by the 26S proteasome complex, and then the release of free and reusable ubiquitin 37 .In vitro studies suggest that overexpression of WT α-Syn 38 or mutant α-Syn 39,40 inhibits proteasome activity in cells, which could impair degradation of protein substrates such as α-Syn.Recently, our group demonstrated that A53T α-Syn overexpression in the rat SNpc leads to catalytic impairment of the 26S proteasome and UPS dysfunction that precedes the loss of dopaminergic neurons and associated motor deficits 12 .Thus, we set out to determine if HFS has any impact on α-Syn-mediated UPS dysfunction.

High frequency stimulation partially rescues α-Syn-mediated autophagy impairment
Macroautophagy is one of the major degradation pathways in cells and plays a pivotal role in maintaining effective turnover of proteins and damaged organelles.Accumulation of pathological proteins may tax the ALP causing its dysfunction.Mutant forms of α-Syn, such as A53T and A30P, bind to lysosomes but are poorly internalized and act as uptake blockers, preventing their own uptake and degradation by lysosomes.This contributes to a vicious cycle of further accumulation 47 and increased autophagic impairment [48][49][50][51] .

α-Syn mediated mitophagy upregulation is reduced by high frequency stimulation
Since HFS partially restores α-Syn-mediated autophagy dysfunction, we explored whether HFS may modulate α-Syn-mediated dysfunction of mitochondrial autophagy.Mitophagy is a process in which damaged or aged mitochondria are selectively removed and degraded via the lysosomal system 58 .Dysregulation of mitophagy has been implicated in PD pathogenesis 59,60 and was recently identified as a potential molecular target of STN-DBS following MPTP administration in mice 62 .The precise role of α-Syn on mitophagy remains poorly defined, with some studies demonstrating that α-Syn can inhibit mitophagy 59,[61][62][63] and others revealing its capacity to induce mitophagy [64][65][66] .We recently demonstrated that overexpression of A53T α-Syn leads to increased mitophagy in SH-SY5Y cells, primary cortical neurons, and in vivo.Further, we found that this phenomenon precedes dopaminergic degeneration 67 , highlighting the importance of restoring mitophagy imbalance to mitigate neurodegeneration 68 .

High frequency electrical stimulation reduces α-Syn levels and α-Syn-mediated autophagy dysfunction in neurons
Having demonstrated the effects of HFS on α-Syn levels and on α-Syn-mediated dysfunction in SH-SY5Y cells, we aimed to replicate key findings in neurons.To achieve this, we cultured primary rat cortical neurons, transduced them with A53T α-Syn or empty vector (EV) AAVs, and used the same HFS parameters 32 .Neurons were co-transduced with YFP as an internal control to assess for non-specific effects of HFS.Corroborating our findings in SH-SY5Y cells, HFS decreased fluorescence levels of A53T α-Syn in stimulated neurons compared to non-stimulated A53T α-Syn transduced neurons (56.38 ± 6.2 RFU stimulated vs 74.0 ± 5.0 RFU non-stimulated, *p < 0.05, one-way ANOVA, Tukey's m.c.t.) (Fig. 7a,b) with no differences in YFP fluorescence intensity observed (118.4 ± 8.3 RFU stimulated vs 131.8 ± 10.4 RFU non-stimulated) (Fig. 7c).

Discussion
Our results illustrate that HFS, mimicking DBS, limits the accumulation of mutant A53T α-Syn and its derangements on cellular pathways involved in the clearance of misfolded proteins and damaged organelles.As part of our previous study 32 , we showed that HFS can decrease levels of mutant α-Syn and WT α-Syn oligomers in transduced cortical neurons stimulated for 3 h.However, the pathway responsible for this reduction was not deciphered.Using the same HFS parameters, we found here that reduction in levels of A53T α-Syn in SH-SY5Y cells and in cortical neurons can occur in as little as 2 h.Furthermore, we found this effect to be stable for short periods of time and transient over longer periods.The levels of α-Syn remained lower 3 h after the end of stimulation and then reverted to that of non-stimulated controls 24 h after the end of stimulation.Moreover, there were no differences in α-Syn mRNA levels between stimulated and non-stimulated A53T α-Syn or pcDNA cells, implying that the transient decrease observed was not due to HFS modifying transcription of α-Syn but due to it enhancing clearance of the accumulated protein.
Prior evidence implicates both UPS 88,89 and ALP 5,47,72,73,90 as pathways responsible for α-Syn degradation.Moreover, dysfunction of these pathways is associated with α-Syn accumulation and implicated in the neurodegenerative process in PD 9,12,46,71 .We found that overexpression of A53T α-Syn in SH-SY5Y cells caused an increase in markers associated with UPS and autophagy dysfunction.We also found that a reduction in α-Syn levels due to HFS was associated with improvements in UPS and autophagy dysfunction in both SH-SY5Y cells and neurons.Furthermore, we demonstrated that in both SH-SY5Y cells and neurons HFS led to a decrease of mitophagy, a process that precedes neurodegeneration 67 .Thus, our observations that HFS reduces not only α-Syn, but also dysfunction in UPS, autophagy, and mitophagy, highlight its therapeutic potential in mitigating the detrimental impact of α-Syn on neurons in multiple manners.
To identify the pathways involved in HFS-mediated α-Syn degradation, we employed the use of pharmacological inhibitors.We observed that HFS failed to alter the increased Ub G76V -GFP levels following MG132-induced proteasome impairment.Unlike that observed in A53T α-Syn transfected cells, HFS was unable to mitigate the elevated levels of Ub G76V -GFP in A53T α-Syn cells treated with MG132, despite reduced α-Syn levels with HFS, signalling that HFS clearance of α-Syn was not dependent on the UPS.Similarly, the autophagy inhibitor BAF did not inhibit the clearance of α-Syn by HFS, which would potentially discount the ALP as a target for electrical stimulation as well.However, we found that HFS reduces autophagy dysfunction due to BAF treatment alone and thus does impact the ALP.
With HFS reducing autophagy dysfunction due to BAF, we focused our attention on BAF's site of action -ATP6V0C -as a potential mechanism for HFS.We analyzed the expression of ATP6V0C which has previously been shown be decreased in PD patient brains 85 .Blockade of ATP6V0C inhibits the acidification of lysosomes and interferes with the ALP, thus preventing degradation and promoting accumulation of aggregated proteins, such as α-Syn 87,91 .Supporting our hypothesis, we found that cells transfected with A53T α-Syn exhibited lower levels of ATP6V0C that could be partially restored by short-term HFS.Furthermore, HFS also restored levels of ATP6V0C following BAF treatment.Although the mechanism underlying the activation of ATP6V0C is unclear, studies have shown that ATP hydrolysis can be differentially modulated by the frequency of oscillating electric fields of sine waveform 92,93 .Increased ATP hydrolysis by V-ATPase leads to influx of protons across the pump, enabling the acidification of lysosomes and subsequent protein degradation by pH-sensitive proteases.Although the electric fields generated in our study were of square waveform, in which the amplitude alternates at a steady frequency between fixed minimum and maximum values, the potential ability of HFS to stimulate V-ATPase to reduce autophagy dysfunction remains a consideration.In vitro DCS has been shown to influence autophagy and oligomeric/aggregated forms of α-Syn while increasing soluble monomeric α-Syn; the most robust effect was observed 17 h from end of stimulation 33 .Notwithstanding the differences in stimulation protocols and experimental parameters, ours is the first study to implicate V-ATPase, specifically ATP6V0C, as a potential site of action for electrical stimulation in models of α-Syn accumulation and α-Syn-mediated cellular dysfunction.
In our study, electrical stimulation caused significant reduction of accumulated α-Syn, but the reduction was not complete and was transient.Electrical stimulation also only partially rescued UPS and autophagy dysfunction.Further research to understand the optimal frequency, pulse width, and current settings sufficient for robust and prolonged reduction in α-Syn and α-Syn-mediated UPS and autophagy impairment is needed.
Our study is not without limitations.Although we propose that HFS targets ATP6V0C in SH-SY5Y cells, the ability of HFS to mitigate the altered levels of ATP6V0C associated with A53T α-Syn overexpression was not observed in neurons, despite ATP6V0C levels being reduced with HFS in EV conditions.Mutant α-Syn overexpression in neurons likely affects more than just the ATP6V0C subunit.Further exploration of other ATPase subunits is needed to confirm the specificity of the action of HFS.Moreover, it is important to note our study involves short-term stimulation and only mutant A53T α-Syn.As such, future studies will be required to assess the mechanistic underpinnings of these changes in other forms of α-Syn (e.g., WT, oligomers, fibrils).Although recapitulating DBS using HFS in vitro to understand the effects on α-Syn and associated cellular dysfunction is feasible, future preclinical studies will be needed to create effective translational paths to explore repurposing DBS for people with PD.
The University Health Network Animal Care Committee approved all animal procedures in accordance with the guidelines and regulations set by the Canadian Council on Animal Care.Experiments were performed in compliance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.